Project Summary/Abstract Zymtronix is developing a series of highly tunable materials and processes for universal enzyme immobilization based on hierarchical magnetic metamaterials. This enzyme immobilization platform is being designed to quickly find the optimal conditions to immobilize single and full systems of enzymes. It affords enzyme stability, maximal use of substrates (including co-factors), and recent developments imparts modularity for flow processes. For this phase I project, Zymtronix proposes to develop its technology to improve the efficiency of defined glycan production ? focusing on human milk oligosaccharides (HMOs)?with optimized full enzyme systems immobilized on macroporous scaffolds specifically designed for continuous flow processing. Libraries of large and complex glycans have been made available, thanks to NIH?s Common Fund program ?Accelerating Translation of Glycoscience: Integration and Accessibility? launched in 2015. Currently, the primary hurdle towards the broader application of complex synthetic glycans in research, preclinical and commercial applications is their availability and affordability. Zymtronix?s technology allows maintaining high activities and co-immobilizing multiple enzymes to mimic live-cell reactions and combined in situ production of active sugars, transfer of the sugar for de novo synthesis, while ensuring the reuse of the enzymes and sub- stoichiometric use of cofactors for significant cost-reduction. For this Phase I we will demonstrate key steps for the production of Human milk oligosaccharides (HMOs), the third largest component of breast milk that are particularly commercially relevant glycan models. While some simple probiotic HMOs can be effectively produced via fermentation for infant formula (2?FL, 3 DP), complex and branched prophylactic HMOs are elusive and have been marginally produced at high cost via chemocatalysis. Zymtronix?s technology solution will significantly improve HMO production efficiency while imparting significant cost savings. The Phase I proof- of-concept will primarily focus on 2 key steps of glycan functionalization with fucose and sialic acid activation and transfer onto HMO backbones with immobilized enzymes. Specific Aims for this Phase I project include 1) Produce two key activated sugars CMP-Sialic acid and GDP-L-fucose with immobilized enzymes; 2) Perform the fucosylation and siallyation of lactose and lactose-N-tetraose with immobilized enzymes; 3) Demonstrate fucosylation of Lactose directly with L-fucose with an immobilized three-enzyme system including GTP recycling. For Phase II, we proposed to expand our continuous flow approach for gram scale synthesis of a glycan library (>4DP) consisting of 16 linear, 38 biantennary, and 6 triantennary HMOs and go beyond HMOs with glycosylation of active small molecules.